Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin

Makvandi‐Nejad, Shokouh; Sheedy, Claudia; Veldhuis, Linda J; Richard, Gabrielle; Hall, J. Christopher

doi:10.1016/j.jim.2010.06.015

Cited by 23 publications

(21 citation statements)

References 23 publications

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“…The quality of the V H H library was estimated based on library size, insert ratio, and diversity of the V H H sequences. The V H H-displayed phage library was amplified and precipitated as described previously [40] with the exception of using helper phage M13K07 (New Englad Biolabs). For round 1 of panning, one well of a microtitre plate was coated with PBS (100 µL) and a second well was coated with 40 µg of α–Cbtx (100 µL).…”

Section: Methodsmentioning

confidence: 99%

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In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

et al. 2013

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Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.

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Section: Methodsmentioning

confidence: 99%

“…Bound phage were eluted with 200 µL of 100 mM triethylamine (TEA) and neutralized with 400 µL of 1 M tris-HCl (pH 7.4). Eluted phage were rescued, amplified and precipitated as described previously [40], and used in preparation for the next round of panning.…”

Section: Methodsmentioning

confidence: 99%

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

et al. 2013

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“…Immune libraries derived from immunized or convalescent animals contain abundant and mature antibodies; thus, they are useful for screening for functional antibodies (9,21). Therefore, we constructed immune libraries using the prokaryotic expression vector pOPE101, and the scFv gene that was derived from the spleens and PBLs of immunized chickens.…”

Section: Discussionmentioning

confidence: 99%

Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus

Lin

et al. 2015

Viral Immunology

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Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression.

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“…The temperature at which phages are produced in Escherichia coli ( E. coli ) is presumed to be the key to achieving this. Previously, an attempt was made to acquire rVH binders using an rVH-displaying phage library produced at 25 °C 16 , which is lower than the conventional temperature for rabbit scFv-displaying phages (30 °C or 37 °C) 17–19 , resulting in obtainment of only weak binders. Thus, in this study, the temperature was further lowered to investigate its impact on the rVH display level and resulting acquisition of rVH binders.…”

Section: Introductionmentioning

confidence: 99%

Efficient generation of single domain antibodies with high affinities and enhanced thermal stabilities

Shinozaki

Hashimoto

Fukui

et al. 2017

Sci Rep

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Single domain antibodies (sdAbs), made of natural single variable regions of camelid or cartilaginous fish antibodies, or unpaired variable regions of mouse or human IgGs, are some of the more promising biologic modalities. However, such conventional sdAbs have difficulties of either using unwieldy animals for immunization or having high affinity deficiencies. Herein, we offer a versatile method to generate rabbit variable domain of heavy chain (rVH) derived sdAbs with high affinities (K D values of single digit nM or less) and enhanced thermal stabilities (equal to or even higher than those of camelid derived sdAbs). It was found that a variety of rVH binders, including those with high affinities, were efficiently acquired using an rVH-displaying phage library produced at a low temperature of 16 °C. By a simple method to introduce an additional disulfide bond, their unfolding temperatures were increased by more than 20 °C without severe loss of binding affinity. Differential scanning calorimetry analysis suggested that this highly efficient thermal stabilization was mainly attributed to the entropic contribution and unique thermodynamic character of the rVHs.

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Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin

Cited by 23 publications

References 23 publications

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus

Efficient generation of single domain antibodies with high affinities and enhanced thermal stabilities

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